The airway epithelium functions as the first-line of defense against inhaled environmental toxins and allergens by facilitating responses to such stimuli. Irregularities of such defensive responses are thought to contribute to chronic respiratory diseases including asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, and lung cancer. The tyrosine kinases Src and epidermal growth factor receptor (EGFR) play a central role in these responses, and promotes signaling cascades that regulate innate epithelial wound healing and inflammation. Moreover, increased Src/EGFR activation also contributes to chronic lung diseases such as asthma. A key component of airway epithelial response to diverse environmental stimuli is activation of the NADPH oxidase, dual oxidase 1 (DUOX1), to generate H2O2, which has recently emerged as essential in signaling pathways by activating signaling proteins. Moreover, our recent studies established that DUOX1 mediates the transactivation of EGFR via initial activation Src, and DUOX1-dependent activation of Src and EGFR are important in innate responses to airborne allergens and the development of allergic asthma. However, the precise mechanisms by which DUOX1-derived H2O2 regulates Src and/or EGFR activation remain unclear. We hypothesize that DUOX1-produced hydrogen peroxide activates Src and EGFR by the oxidation of critical protein cysteine residues to stimulate innate airway epithelial repair and immune responses. To address this hypothesis, we will first use purified EGFR and Src to determine H2O2-dependent mechanisms of kinase activation by evaluating cysteine oxidation and tyrosine phosphorylation using a combination of immunological and analytical approaches. We will use mass spectrometry to identify the precise cysteine(s) oxidized by H2O2 and protein structures to model the chemical mechanism(s) whereby H2O2 causes activation of Src and EGFR. In the second stage, we will use site-directed mutagenesis to determine the effect of removing critical cysteine residues within Src to determine their involvement in epithelial wound repair and immune responses. Modulations of cellular migration and allergic response with a redox-insensitive Src will assess the significance of oxidative activation in these processes in addition to providing insights into the effect of Src protein structure on epithelial function. This strategy involves an innovative approach toward understanding cell signaling on a molecular level, and completion of this research will reveal important details into redox- dependent signaling events that directly affect epithelial defense in health and disease.